APOE (Apolipoprotein E) is part of APOE/APOC gene cluster on chromosome 19 and codes for 3 protein isoforms ? APOE2, APOE3 and APOE4. APOE transports cholesterol and phospholipids in the periphery and brain1. APOE isoforms differ in their lipid and receptor binding capacity and their role is associated with clearance of LDL, VLDL and chylomicrons. The inheritance of APOE4 allele increases and APOE2 decreases the risk for late onset AD (LOAD), but the mechanism is poorly understood. While APOE is involved in critical cellular functions such as oxidative processes, inflammation, glial cell and neuronal homeostasis, none of those can be dissociated from isoform specific binding, transport and delivery of cholesterol and phospholipids to different cell types. Recent reports suggest there might be a differential APOE-isoform specific effect on microglia mediated phagocytosis and function of immune receptors expressed in brain16. Our preliminary data demonstrate APOE isoforms influence expression of immune receptors Dectin-1/Clec7a, Siglec1, Siglech, Oscar - involved in inflammatory response and phagocytosis. A strong support to an interconnected role for APOE and immune receptor mediated phagocytosis are our results of Multi-Dimensional Mass Spectrometry Shotgun Lipidomics of brain samples from AD patients, where we find significant differences in phospholipid molecular speciation in major phospholipid classes. We hypothesize the APOE isoform-specific effects on phagocytosis are driven by the different phospholipid composition of APOE lipid particles and/or by the differential effect of APOE isoforms on microglial transcriptome. We are proposing 3 Specific Aims to test the hypothesis: Aim 1. Determine age related APOE isoform-specific effect on molecular phospholipid profiling in brain: we will perform lipidomics assays on phospholipid content and molecular speciation of APOE2, APOE3 and APOE4 lipid particles in Astrocyte Conditioned Medium, Interstitial Fluid and brain parenchyma of WT and APP expressing mice with targeted APOE replacement at two ages, and in brain samples from nondemented control individuals and AD patients. Aim 2. Establish the role of APOE isoforms for immune receptor mediated Ab phagocytosis: we will determine if phospholipid molecular species part of native APOE particles secreted by astrocytes, or derived from brain Interstitial Fluid (ISF) differentially affect microglial/immune receptor mediated phagocytosis. Aim 3. In APOE targeted replacement mice to establish APOE isoform? and age?specific response to HFD and its impact on CNS transcriptome: The objectives are to perform behavioral, integrated transcriptomics, and co-expression network analysis on microglia from brain samples of WT and APP mice expressing human APOE isoforms.